Tetrafluorobenzyl derivatives and pharmaceutical composition for preventing and treating acute and chronic neurodegenerative diseases in central nervous system containing the same

ABSTRACT

The present invention relates to a tetrafluorobenzyl derivative and a pharmaceutical composition for prevention and treatment of acute and chronic neurodegenerative disease in central nervous system and ophthalmic diseases containing the same. The tetrafluorobenzyl derivative of the present invention can effectively be used to prevent and treat chronic neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease and Huntington&#39;s disease, degenerative brain disease such as epilepsy and ischemic brain disease such as stroke.

TECHNICAL FIELD

The present invention relates to a tetrafluorobenzyl derivative and apharmaceutical composition comprising the same as a pharmaceuticallyeffective ingredient and, more particularly, to a noveltetrafluorobenzyl derivative therapeutically effective for the treatmentand prevention of neurological diseases and ocular diseases.

BACKGROUND ART

Recent advances in medicine have extended the life span of human beingsand as a result, age-related acute and chronic neurological diseases,such as Alzheimer's disease, stroke, Parkinson's disease etc. increase.These neurological diseases are characterized by the progress ofdegeneration of specific neurons over the course of diseases. As matureneurons do not regenerate once they die, neuronal death in neurologicaldiseases above can result in incurable loss of essential brain functionincluding cognition, sensation, and movement and thus economic andsocial overload.

Exicitotoxicity, oxidative stress, and apoptosis have been implicated asmajor routes of neuronal death occurring in various neurologicaldiseases and propagate through distinctive signaling pathways for eachroute.

Glutamate is the excitatory neurotransmitter mediating slow excitatorysynaptic transmission through N-methyl-D-aspartate (NMDA) receptors andfast excitatory synaptic transmission through kainate orα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptors. In theresting state of neurons, Mg²+blocks NMDA receptor channels in avoltage-dependent manner. With stimuli causing membrane depolarization,Mg²⁺ is liberated from the NMDA channels, rendering the channelspermeable to Ca²⁺ and Na⁺. Activation of NMDA receptors plays animportant role in physiological process including learning and memory[Siegel G. J. et al., Basic Neurochemistry, 6^(th) edition, LippincottWilliams & Wilkins, 315-333 (1999)]. Besides physiological roles, briefand excess activation of NMDA receptors can cause rapidly evolvingneuronal death and mechanisms underlying NMDA receptor-mediatedneurotoxicity have been extensively studied over the last tow decades.

In 1969, Olney et al. reported that oral administration of monodiumglutamate produced neuronal cell death in brain of mice or monkey[Olney, J. W. and Sharpe, L. G., Science, 166:386-388(1969); Olney, J.W. and Ho, O. L., Nature, 227(258): 609-611(1970)], suggesting thatglutamate, the excitatory neurotransmitter, mediates neuronalexcitability and death in epilepsy [Olney, J. W., Int. Rev. Neurobiol.,27:337-62:337-362(1985)]. Administration of glutamate induces neuronaldeath in cultured cortical neurons, which occurs through activation ofNDMA receptors and depends upon Ca²⁺ entry [Choi, D. W., J. Neurosci.,7(2)369-379(1987)]. Glutamate neurotoxicity (or excitotoxicity) has beenproposed as a main pathway to neuronal death in stroke as well asepilepsy [Choi. D. W., Neuron, 1:623-634(1988)]. Interrupted bloodsupply to brain results in deprivation of oxygen and glucose, whichcauses energy (ATP) failure, dysfunction of ATP-dependent ion channels,an d membrane depolarization that increases glutamate release. Energyfailure also reduces glutamate uptake into glial cells. Consequently,glutamate is abnormally accumulated in the synaptic cleft [Choi, D. W.and Rothman, S. M., Annu. Rev. Neurosci., 13:171-182(1990); Benveniste,H et al., J. Neurochem., 43(5):1369-1374(1984)]. The excess accumulationof glutamate causes neuronal cell death primarily through activation ofNMDA receptors. In fact, administration of NMDA receptor antagonistshave been reported to reduce neuronal death following hypoxic-ischemicbrain injury [Goldberg, M. P. et al., J. Pharmac. Exp. Ther.,243:784-791(1987); Simon et al., Science 226:850-852 (1984); Sheardown,M. J. et al., Science 247:571-574(1990)].

Extensive evidence supports that excitotoxicity also contributes toneuronal death in neurodegenerative diseases. The key pathologicalfeatures of Huntington's disease (HD) include degeneration of GABAergicneurons and selective sparing of NADPH diaphorase-containing neurons inthe striatal area. These pathological features of HD are observedfollowing the intrastriatal injections of NMDA or quinolinic acid, anNMDA receptor agonist [Ferrante, R. J et al., Science,230(4625):561-563(1985); Beal, M. F. et al., Nature,321(6066):168-171(1986); Koh, J.Y. et al., Science,234(4772):73-76(1986)]. Amytrophic lateral sclerosis (ALS) isaccompanied by degeneration of upper and lower motor neurons and markedby neurogenic atrophy, weakness, and fasciculation. While thepathogenesis of ALS remains to be resolved, excitotoxicity has beenexpected to participate in the process of the ALS. In particular, ALSpatients show defects in synthesis and transport of glutamate andincreased levels of extracellular glutamate [Rothstein, J. D., Clin.Neurosci., 3(6):348-359(1995); Shaw, P. J. and Ince, P. G., J. Neurol.,244 Suppl 2:S3-14(1997)].

Although NMDA receptor-mediated excitotoxicity plays a causative role instroke and neurodegenerative diseases, the therapeutic potential of NDMAreceptor antagonists has been limited by unexpected side effects inbrain. In particular, systemic administration of NMDA receptorantagonists impairs normal brain function and can cause widespreadneuronal damage in adult rat brain [Olney et al., Science 244:1360-1362(1989)]. The neuropsychopathological side effects are produced byhigh-affinity NMDA receptor antagonists such as phencyclidine andrelated NMDA receptor antagosints such as MK-801 (dizocilpine maltate),tiletamine and ketamine and may be overcome with administration ofchannel-blocking NMDA receptor antagonists with low affinity andrapid-kinetic response [Rogawski, Amino Acids 19:133-149 (2000)].

Free radicals mediate neuronal death occurring in neurological diseasesas well as tissue damage occurring in the whole body [Halliwell, B. andGutteridge, J. M., Mol. Aspects. Med., 8(2):89-193(1985); Siesjo, B. K.et al., Cerebrovasc. Brain Metab. Rev., 1(3):165-211(1989); Schapira, A.H., Curr. Opin. Neurol., 9(4)260-264(1996)]. Free radicals are producedin degenerating brain areas following hypoxic-ischemia or traumaticbrain and spinal cord injuries. Antioxidants or maneuvers scavengingfree radicals attenuate brain damages by hypoxic-ischemia or traumaticinjuries [Flamm, E. S. et al., Stroke, 9(5):445-447(1978); Kogure, K. etal., Prog. Brain Res., 63:237-259(1985); Chan, P. H. J. Neurotrauma., 9Suppl 2:S417-423(1992); Faden, Pharmacol.Toxicol. 78:12-17 (1996)].Extensive evidence supports that free radicals are produced in brainareas undergoing degeneration in neurodegenerative diseases possibly dueto point mutations in Cu/Zn superoxide dismutase in ALS [Rosen et al.,Nature 362:59-62 (1993)], the decrease of reduced glutathione,glutathione peroxidase, and catalase, and the increase of iron insubstatia nigra in Parkinson's disease [Sofic, E. et al., J. NeuralTransm., 74:199-205(1988); Fahn, S. and Cohen, G., Ann. Neurol.,32(6):804-812(1992)], the oxidation of lipid, nucleotides, and protein,an increase of iron in degenerating neural tissues, and generation offree radicals by beta amyloid in Alzheimer's disease brain [Schubert, D.et al., Proc. Natl. Acad. Sci. U.S.A. 92(6):1989-1993(1995); Richardson,J. S. et al., Ann. N. Y. Acad. Sci. 777:362-367(1996)], andmitochondrial dysfunction in HD [Dexter, D. T. et al., Ann Neurol. 32Suppl:S94-100(1992)). Accordingly, antioxidants have beenneuroprotective against such neurodegenerative diseases [Jenner, Pathol.Biol. (Paris.) 44:57-64 (1996); Beal, Ann. Neurol. 38:357-366 (1995)].

Zinc (Zn²⁺) is a transition metal which is highly present and plays adynamic role in brain. Within cells, zinc is associated withmetalloproteins to control the enzymatic activity and structuralstability of the proteins. Also, zinc regulates gene expression bybinding to various transcription factors. In the CNS, zinc is localizedat the synaptic terminal of glutamatergic neurons, released in anactivity-dependent manner, and regulates activity of variousneurotransmitter receptors and ion channels.

Zn²⁺ mediates neurodegenerative process observed in seizure, ischemia,trauma, and Alzheimer's disease (AD). The central administration ofkainate, a seizure-inducing excitotoxin, causes the translocation ofZn²⁺ into postsynaptic degenerating neurons in several forebrain areas.Translocation of zinc into adjacent neurons was also observed followingischemic and traumatic brain disease, and the blockade of its transitioninhibited neuronal cell death [Frederickson, C. J. and Bush, A. I.,Biometals. 14:353-366(2001); Weiss et al., Trend. Pharmacol. Sci.21:395-401(2001)]. Zinc has been known to enter neurons through Ca²⁺permeable NMDA and AMPA/KA receptors, voltage-gated Ca²⁺ channel, orzinc transporter protein, and to induce neuronal death by the activationof NADPH oxidase generating reactive oxygen species. Zn²⁺ is observed inthe extracellular plaque and degenerating neurons in AD, which likelycontributes to neuronal degeneration in AD [Suh et al., Brain Res.852:274-278(2000); Bush et al., Science 265:1464-1467 (1994); Lee etal., Proc. Natl. acad. Sci. U.S.A. 99:7705-7710(2002)]. Therefore, theinhibition of release and toxicity of zinc has been suggested as newstrategy of prevention and treatment for Alzheimer's disease[Fredrickson and Bush, Biometals;, 14:353-66(2001)].

As described above, NMDA receptor-mediated excitotoxicity, oxidativestress, and zinc can contribute to neuronal death in various acute andneurodegenerative diseases in the nervous system. Thus, efficienttherapeutic drugs preventing each route of neuronal deaths should bedeveloped to treat such catastrophic neurological diseases.

We have investigated to develop neuroprotective drugs with multipleneuroprotective effects against excitotoxicity or oxidative stress andsucceeded in inventing tetrafluorobenzyl derivatives that can be appliedto treat stroke, trauma, and some neurodegenerative diseases.

DISCLOSURE OF THE INVENTION

Leading to the present invention, the intensive and thorough research onthe treatment of disorders in the central nervous system, conducted bythe present inventors, results in the finding that noveltetrafluorobenzyl derivatives have potent neuroprotective activityagainst various types of neuronal death induced in cell culture andanimal models of neurological diseases.

Accordingly, it is an object of the present invention to provide a noveltetrafluorobenzyl derivative.

It is another object of the present invention to provide apharmaceutically effective composition for the treatment and preventionof neurological diseases and ocular diseases.

In one aspect of the present invention, there is provided a noveltetrafluorobenzyl derivative, represented by the following chemicalformula 1,

wherein,

R₁, R₂, and R₃ are hydrogen or halogen;

R₄ is hydroxy, alkyl, alkoxy, halogen, alkoxy substituted with halogen,alkanoyloxy or nitro;

R₅ is carboxylic acid, ester of carboxylic acid substituted with C₁-C₄alkyl, carboxyamide, sulfonic acid, halogen, or nitro;

In another aspect of the present invention, there is provided apharmaceutical composition for the prevention and treatment ofneurological diseases and ocular diseases, comprising thetetrafluorobenzyl derivative or its pharmaceutically acceptable salt asan effective ingredient.

The present invention provides a novel tetrafluorobenzyl derivativerepresented by the following chemical formula 1, or its pharmaceuticallyacceptable salt.

wherein,

R₁, R₂, and R₃ are hydrogen or halogen

R₄ is hydroxy, alkyl, alkoxy, halogen, alkoxy substituted with halogen,alkanoyloxy or nitro;

R₅ is carboxylic acid, ester of carboxylic acid substituted with C₁-C₄alkyl, carboxyamide, sulfonic acid, halogen, or nitro;

In here, alkyl group is C₁-C₄ alkyl and more preferably C₁-C₂ alkyl.Alkyl described above definitely contains methyl, ethyl, propyl,isopropyl, n-butyl, sec-butyl, or tert-butyl.

Alkoxy group is C₁-C₄ and more preferably C₁-C₂ alkoxy. Alkoxy describedabove definitely contains methoxy, ethoxy, or propanoxy.

Halogen can be substituted with fluoride, chloride, bromide, or iodide.

Alkanoyloxy is C₂-C₁₀ alkanoyloxy and more preferably C₃-C₅ alkanoyloxy.Alkanoyloxy described above definitely contains ethanoyloxy,propanoyloxy, or cyclohexanecarbonyloxy.

In ester of carboxylic acid, carbon can be substituted with methyl,ethyl, isopropyl, or butyl.

Specific compounds of interest within Formula I are as follows:

2-Hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoicacid (hereinafter, referred to as ‘2-Hydroxy-TTBA’),

2-Nitro-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid,

2-Chloro-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid,

2-Bromo-5-(2,3,5,6-tetrafluoro-4-triflubromethylbenzylamino)benzoicacid,

2-Hydroxy-5-(2,3,5,6-tetrafluoro-4-methylbenzylamino)benzoic acid,

2-Methyl-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid,

2-Methoxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid,

5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)-2-trifluoromethoxybenzoic acid.

2-Nitro-4-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)phenol,

2-Chloro-4-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)phenol(hereinafter, referred to as ‘2-Chloro-TTP’),

2-Hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)Benz amide(hereinafter, referred to as ‘2-Hydroxy-TTA’),

2-Hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzenesulfonic acid (hereinafter, referred to as ‘2-Hydroxy-TTS’),

Methyl2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoate,

2-Ethanoyloxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid (hereinafter, referred to as ‘2-Ethan-TTBA’),

2-Propanoyloxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid (hereinafter, referred to as ‘2-Propan-TTBA’), or

2-Cyclohexanecarbonyloxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid (hereinafter, referred to as ‘2-Cyclohexan-TTBA’).

However, the compounds described above are just representative of thepresent invention, which could include more compounds.

The present invention provides tetrafluorobenzyl derivatives representedby formula (I) and pharmaceutical composition for prevention andtreatment of neurological and ocular diseases containing the effectivecomponent as a pharmaceutically acceptable salt.

As drugs for pharmaceutical use, the salt of the compound of formula (I)doesn't have toxicity, and should be pharmaceutically acceptable.Various kinds of salts can be used to prepare pharmaceuticallyacceptable salts, including non-toxic compound of the present invention.

The pharmaceutically acceptable salts of the compounds in the presentinvention include alkali metals, such as lithium, sodium or potassium,and alkaline earth metals, such as calcium or magnesium. Acid additionsalts may be prepared by reacting the solution of pharmaceuticallyacceptable nontoxic salts such as hydrochloric acid, fumaric acid,maleic acid, succinic acid, acetic acid, citric acid, tartaric acid,carbonic acid, or phosphoric acid with the compound of the invention.

The compound of formula (I) in the present invention can be used forcure of normal and pathological neurodegenerative diseases amongdiseases or symptoms in cerebrovascular and neurological systems. In theconcrete, the compound above represented by formula (I) is used forprevention or treatment of thromboembolism, ischemic stroke, hemorrhagicstroke, cerebrovascular convulsion, brain aging, traumatic brain injury,traumatic spinal cord injury, cardiac arrest, arterial hypotention,hypoglycemia, anoxia, and hypoxia. Also, the compound of formula (I) inthe present invention can be beneficially used for decreasingneurodegenerative diseases such as Huntington's disease, Alzheimer'sdisease, senile dementia, Pick's disease, Korsakov's syndrome,olivopontocerebellar degeneration, amyotrophic lateral sclerosis (ALS),Parkinson's disease, Down's syndrome, Glutaric acidaemia, epilepsy,multi-infarct dementia, and brain inflammation. They have application totreatment of ocular diseases such as glaucoma, macular degeneration,diabetic retinopathy, uveitis. Moreover, they have application to theprevention and treatment of drug addiction, depression, and pain.

The composition of the present invention can be treated by oraladministration, intravenous injection or non-oral administration, andtreated by various forms such as tablet, capsule, powder, grain,sterilized solution, suspension or suppository for rectaladministration. Major effective elements of the composition can be madeas a solid tablet using pharmaceutical carriers, for example commontablet element such as corn dextrin, lactose, sucrose, sorbitol, talc,stearic acid, magnesium stearate, decalcium phosphate or gums, andadditional pharmaceutical diluted solution. Tablets or pellets of thepharmaceutical composition in the present invention can be manufacturedfor sustained release dosage form as facilitated forms foradministration using well-known coating method etc. in the appropriateindustry. For example, tablets or pellets can be composed with inner andouter administrative elements. The inner administrative elements oftablets or pellets can be manufactured as wrapped with outeradministrative elements. Liquid forms of the composition in the presentinvention manufactured for oral administration or the injection includesolution, appropriately flavored syrup, water-soluble suspension,water-insoluble suspension, emulsion made by edible oil such as cottonoil, sesame oil, coconut oil, or peanut oil, elixir, and similarpharmaceutical carriers. Tragacanth gum, acacia, alginic acid sodiumsalt, dextran, sodium carboxymethylcellulose, methylcellulose,polyvinylpyrrolidone, or synthesized or natural gums like gelatin etccan be used as appropriated aid to dispersion or suspension in makingwater-soluble suspension.

Quantity of medication can be determined by several related factors suchas diseases, age, sex, weight, and degrees of illness of patients etc.for the treatment of neurodegeneration.

The tetrafluorobenzyl derivatives related to the present invention maybe synthesized from the following reaction schemes. However, thecompounds described in the schemes are just representative of thepresent invention, which could include more compounds.

The tetrafluorobenzyl derivatives were synthesized from the followingreaction. First, the nitrobenzene compounds, where hydrogens at 3 and 4positions were substituted with R₅ and R₄ respectively, werehydrogenated for 12 hours under 3 atm pressure (reaction condition a).The resulting aniline compounds were reacted with2,3,5,6-tetrafluoro-4-methylbenzyl bromide in DMF in the presence oftriethylamine for 12 hours to give the desired tetrafluorobenzylderivatives (reaction condition b).

In the above scheme, R₁, R₂, and R₃ represent hydrogen or halogen; R₄represents hydroxy, alkyl, alkoxy, halogen, alkoxy substituted withhalogen, alkanoyloxy, nitro; R₅ represents carboxylic acid, esters ofcarboxylic acid, carboxyamide, sulfonic acid, halogen, and nitro group.

For the synthesis of compound where R₄ is hydroxy group and R₅ iscarboxyamide group, the hydroxy and amino group in5-(2,3,5,6-tetrafluoro-4-trifluorobenzylamino)benzoic acid were firstprotected with trifluoro group by reacting with trifluoromethyl aceticanhydride in the catalytic amount of c-H₂SO₄ (reaction condition a). Theresulting2-carboxy-5-[2,3,5,6-tetrafluoro-4-trifluoromethyl-benzyl]-(2,2,2-trifluoroacetyl)amino]phenylester compound was then reacted with SOCl₂ followed by ammoniumcarbonate to give2-carbamoyl-4-[2,3,5,6-tetrafluoro-4-trifluoromethyl-benzyl-(2,2,2-trifluoroacetyl)amino]phenylester compound (reaction condition b), which was then hydrolyzed withHCl solution to give the desired carboxyamide compound. (reactioncondition c) In this scheme, R₁, R₂, R₃ and R₄ are same groups, whichare previously defined while R₅ are C₁-C₄ substituted alkyl group.

SYNTHESIS EXAMPLE 1 Preparation of2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid (2-Hydroxy-TTBA)

To a solution of 5-aminosalicylic acid (1.02 g, 6.66 mmole, purchasedfrom Aldrich Chemical Company, USA, A7, 980-9) and triethylamine (1 ml)in dried DMF (80 ml) was added2,3,5,6-tetrafluoro-4-trifloromethylbenzyl bromide (1.23 g, 7.18 mmole)(Aldrich, 40, 640-6) at room temperature under a nitrogen atmosphere.The reaction mixture was stirred for 2 hr at room temperature and thensolvent was removed in vacuo. The reaction mixture was diluted withethyl acetate and then extracted with ethyl acetate. The organic layerwas washed with water and brine, and then dried over anhydrous MgSO₄.After evaporation of the solvent, the residue was recrystallized fromether/hexane (1:10) to give 1.60 g (64% yield) of2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid as a white solid.

mp 179° C.,

¹H-NMR: 8.0 (s, 1H), 7.3 (d, 1H), 6.7 (t, 1H), 5.5 (s, 2H),

IR(KBr pellet): 3386, 1741, 1500 cm⁻¹ Elemental analysis for C₁₅H₈F₇NO₃% C % H % N % F % O Calculated 47.01 2.10 3.66 34.70 12.53 Found 47.002.03 3.69

SYNTHESIS EXAMPLE 2 Preparation of2-nitro-5-(2,3,5,6-tetrafluoro-4-trifluoromethy benzylamino)benzoic acid

According to the similar procedure in Synthesis Example 1, by using5-amino-2-nitrobenzoic-acid (1.03 g, 5.65 mmole) and2,3,5,6-tetrafluoro-4-trifloromethylbenzyl bromide (1.01 g, 6.04mmole)(ACROS, 33074-0010), 1.50 g (76.3% yield) of2-nitro-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoic acidwas obtained as a pale yellow solid.

mp 121° C.,

¹H-NMR: 8.0 (d, 1H), 7.3 (s, 1H), 6.7 (d, 1H), 5.5 (s, 2H),

IR(KBr pellet): 3417, 1703, 1504 cm⁻¹ Elemental analysis for C₁₅H₇F₇N₂O₄% C % H % N % F % O Calculated 43.71 1.71 6.80 32.36 15.53 Found 43.371.68 6.50

SYNTHESIS EXAMPLE 3 Preparation of2-chloro-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid

According to the similar procedure in Synthesis Example 1, by using5-amino-2-chlorobenzoic acid (1.02 g, 5.94 mmole)(ACROS, 32525-5000),DMF (50 ml) and 2,3,5,6-tetrafluoro-4-trifloromethylbenzyl bromide (1.16g, 6.99 mmole), 2.04 g (85.5% yield) of2-chloro-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino) benzoicacid was obtained as a pale brown solid.

mp 58° C.,

¹H-NMR: 7.26 (d, 1H), 7.24 (s, 1H), 6.7 (d, 1H), 4.12 (s, 2H),

IR(KBr pellet): 3402, 1720, 1494, 1434; 929 cm⁻¹

HPLC (0.01% TFA-ethyl acetate 0.1 TFA-water=80:20, Rt=4.2 mins): 97%purity Elemental analysis for C₁₅H₇ClF₇NO₄ % C % H % N % F % OCalculated 44.85 1.76 3.49 33.11 7.97 Found 44.83 2.31 3.43

SYNTHESIS EXAMPLE 4 Preparation of2-bromo-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoic acid(4-1) Preparation of 5-amino-2-bromobenzoic acid

A mixture of 2-bromo-5-nitrobenzoic acid (1.10 g, 4.06 mmol) (Aldrich,38, 184-5), activated Pd-C (43.62 mg, 0.41 mmol)(Aldrich, 20, 569-9) inmethanol (30 ml) was hydrogenated for 4 hr under 30 psi of hydrogenpressure. After the mixture was filtered, the filtrate was concentratedto give 0.80 g (91.2% yield) of 5-amino-2-bromobenzoic acid as a paleyellow solid.

IR(KBr pellet): 3111, 2558, 2499, 1716, 1676 cm⁻¹

(4-2) Preparation of2-bromo-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoic acid

According to the similar procedure in Synthesis Example 1, by using5-amino-2-bromobenzoic acid (1.05 g, 6.12 mmole) which was prepared fromSynthesis Example (4-1), DMF (50 ml) and2,3,5,6-tetrafluoro-4-trifloromethylbenzyl bromide (1.16 g, 6.99 mmole),2.02 g (76.9% yield) of2-bromo-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoic acidwas obtained as a pale yellow solid.

mp 70° C.,

¹H-NMR: 7.42 (d, 1H), 7.3 (s, 1H), 6.9 (d, 1H), 5.5 (s, 2H),

IR(KBr pellet): 3438, 1695, 1491, 1425, 939 cm⁻¹

HPLC (0.1% TFA-ethyl acetate: 0.1% TFA-water=80:20, Rt=4.5 mins) 99%purity Elemental analysis for C₁₅H₇BrF₇NO₂ % C % H % N % Br % F % OCalculated 40.38 1.58 3.14 17.91 29.81 7.17 Found 44.62 1.57 3.79

SYNTHESIS EXAMPLE 5 Preparation of2-hydroxy-5-(2,3,5,6-tetrafluoro-4-methylbenzylamino)benzoic acid

According to the similar procedure in Synthesis Example 1, by using4-methyl-2,3,5,6-tetrafluoro-benzyl bromide (1.23 g, 7.18mmole)(Aldrich, 40, 646-6) instead of2,3,5,6-tetrafluoro-4-trifluoromethyl benzylbromide, 1.60 g (64.0%yield) of 2-hydroxy-5-(2,3,5,6-tetrafluoro-4-methylbenzylamino)benzoicacid was obtained as a white solid.

mp 212° C.,

¹H-NMR: 8.0 (s, 1H), 7.3 (d, 1H), 6.7 (t, 1H), 5.5 (s, 2H), 2.2˜2.3 (s,3H),

IR(KBr pellet): 3386, 1741, 1500 cm⁻¹ Elemental analysis for C₁₅H₁₁F₄NO₃% C % H % N % F % O Calculated 54.72 3.37 4.25 23.08 14.58 Found 54.903.60 4.06

SYNTHESIS EXAMPLE 6 Preparation of2-methyl-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid (6-1) Preparation of 2-methyl-5-nitrobenzoic acid

To a solution of 2-methylbenzoic acid (3.05 g, 22.3 mmole) (ACROS,13904-0010) in c-HNO₃ (20 ml) was carefully added Conc.H₂SO₄ (15 ml) at0° C. The resulting solution was refluxed at 100-120° C. for 5 hours.After the reaction mixture was cooled to room temperature, 50 ml of icechip was added. The resulting precipitate was filtered, washed withwater and dried to give 3.90 g (97.5% yield) of 2-methyl-5-nitrobenzoicacid as a white solid.

IR(KBr pellet): 1531, 1350 cm⁻¹

(6-2) Preparation of 5-Amino-2-methylbenzoic acid

According to the similar procedure in Synthesis Example (4-1), by using2-methyl-5-nitrobenzoic acid (4.10 g, 22.6 mmole) which was preparedfrom Synthesis Example (6-1), 2.01 g (60.0% yield) of5-amino-2-methylbenzoic acid was obtained as a white solid.

IR(KBr pellet): 3437, 3336, 1633, 1400 cm⁻¹

(6-3) Preparation of2-methyl-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid

According to the similar procedure in Synthesis Example 1, by using5-amino-2-methylbenzoic acid (2.06 g, 15.0 mmole), DMF (60 ml), TEA (4ml), and 2,3,5,6-tetrafluoro-4-trifluoromethylbenzyl bromide (5.52 ml,17.7 mmole), 1.50 g (27.0% yield) of2-methyl-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid was obtained as a yellow solid.

mp 84° C.,

¹H-NMR: 7.32(s, 1H), 7.3(d, 1H), 6.9(d, 1H), 5.5(s, 2H), 2.2(s, 3H),

IR(KBr pellet): 3417, 1716, 1496 cm⁻¹ Elemental analysis for C₁₆H₁₀F₇NO₂% C % H % N % F % O Calculated 50.41 2.64 3.53 33.48 12.08 Found 50.402.60 3.39

SYNTHESIS EXAMPLE 7 Preparation of2-methoxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid (7-1) Preparation of 2-methoxy-5-nitrobenzoic acid

According to the similar procedure in Synthesis Example (6-1), by using2-methoxybenzoic acid (2.10 g, 13.79 mmole) (ACROS, 17375-2000), 2.50 g(97.0% yield) of 2-methoxy-5-nitrobenzoic acid was obtained as a whitesolid.

IR(KBr pellet): 3099, 2986, 2965, 1736, 1547 cm⁻¹

(7-2) Preparation of 5-amino-2-methoxy-benzoic acid

According to the similar procedure in Synthesis Example (4-1), by using2-methoxy-5-nitrobenzoic acid (4.10 g, 22.6 mmole) which was preparedfrom Synthesis Example (7-1), 1.90 g (98.0% yield) of5-amino-2-methoxybenzoic acid was obtained as a brown solid.

IR(KBr pellet): 1394, 1220 cm⁻¹

(7-3) Preparation of2-methoxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid

According to the similar procedure in Synthesis Example 1, by using5-amino-2-methoxy-benzoic acid (2.10 g, 12.7 mmole), DMF (50 ml), TEA (6ml), and 2,3,5,6-tetrafluoro-4-trifluoromethylbenzyl bromide (2.00 ml,14.0 mmole), 1.50 g (31.5% yield) of2-methoxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid was obtained as a yellow solid:

mp 94° C.,

¹H-NMR 7.42(s, 1H), 6.4(d, 1H), 6.2(d, 1H), 5.5(s, 2H), 3.73(s, 3H),

IR(KBr pellet): 3429, 1730, 1496 cm⁻¹ Elemental analysis for C₁₆H₁₀F₇NO₃% C % H % N % F % O Calculated 48.38 2.54 3.53 33.48 12.08 Found 47.502.20 3.39

SYNTHESIS EXAMPLE 8 Preparation of5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-2-trifluoromethoxybenzoicacid (8-1) Preparation of 5-nitro-2-trifluoromethoxybenzoic acid

According to the similar procedure in Synthesis Example (6-1)(Lancaster,15687), by using 2-trifluoromethoxybenzoic acid (2.10 g, 9.81 mmole),1.40 g (55.0% yield) of 5-nitro-2-trifluoromethoxybenzoic acid wasobtained as a white solid.

IR(KBr pellet): 1488, 1354 cm⁻¹

(8-2) Preparation of 5-amino-2-trifluoromethoxybenzoic acid

According to the similar procedure in Synthesis Example (4-1), by using5-nitro-2-trifluoromethoxybenzoic acid (1.40 g, 5.34 mmole) which wasprepared from Synthesis Example (8-1), 1.02 g (90.0% yield) of5-amino-2-trifluoromethoxybenzoic acid was obtained as a pale yellowsolid.

IR(KBr pellet): 1627,1369 cm⁻¹

(8-3) Preparation of5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)-2-trifluoromethoxybenzoicacid

According to the similar procedure in Synthesis Example 1, by using5-amino-2-trifluoromethoxybenzoic acid (1.35 g, 6.23 mmole), DMF (50ml), TEA (6 ml), and 2,3,5,6-tetrafluoro-4-trifluoromethylbenzyl bromide(1.10 ml, 6.73 mmole), 2.25 g (81.0% yield) of5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)-2-trifluoromethoxybenzoicacid was obtained as a yellow solid.

mp 38° C.,

¹H-NMR 7.3 (s, 1H), 7.12 (d, 1H), 6.8 (d, 1H), 5.5 (s, 2H),

IR(KBr pellet): 3383, 1712, 1504, 1446, 929 cm⁻¹ Elemental analysis forC₁₆H₇F₁₀NO₃ % C % H % N % F % O Calculated 42.59 1.56 3.0 42.10 10.64Found 41.24 2.20 3.39

SYNTHESIS EXAMPLE 9 Preparation of2-nitro-4-(2,3,5,6-tetrafluoro-4-trifluorormethylbenzylamino)phenol

According to the similar procedure in Synthesis Example 1, by using4-amino-2-nitrophenol (1.00 g, 6.49 mmole), DMF (30 ml), TEA (0.5 ml),and 2,3,5,6-tetrafluoro-4-trifluoromethylbenzyl bromide (1.30 g, 7.78mmole), 1.00 g (40.0% yield) of2-nitro-4-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)phenol wasobtained as a reddish solid.

mp 126-128° C.,

¹³H-NMR(DMSO-d₆) δ 4.23 (d, 2H), 6.93 (m, 2H), 7.12 (s, 1H),

¹³C NMR(DMSO-d₆) δ 36.36, 105.48, 120.63, 122.64, 123.32, 136.17,140.86, 142.34, 144.06, 144.83, 146.44, 151.23,

IR(neat): 3391, 3255, 1545, 1339 cm⁻¹ Elemental analysis for C₁₄H₇F₇N₂O₃% C % H % N % F % O Calculated 43.65 2.09 7.27 28.46 13.69 Found 43.682.05 7.26

SYNTHESIS EXAMPLE 10 Preparation of2-chloro-4-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)phenol

According to the similar procedure in Synthesis Example 1, by using4-amino-2-chlorophenol (3.00 g, 19.6 mmole), DMF (80 ml), TEA (0.5 ml),and 2,3,5,6-tetrafluoro-4-trifluoromethylbenzyl bromide (1.40 g, 8.36mmole), 2.00 g (76.0% yield) of2-chloro-5-(2,3,5,6-tetrafluoro-4-methylbenzylamino)phenol was obtainedas a yellow solid.

mp 52° C.,

¹H-NMR(CDCl₃) 6.9 (d, 1H), 6.7 (s, 1H), 6.5 (d, 1H), 4.4 (s, 2H),

IR(KBr pellet): 3382, 1687, 1617, 1586 cm⁻¹ Elemental analysis forC₁₄H₇F₇N₂O₃ % C % H % N % Cl % F % O Calculated 42.00 1.89 3.75 9.4935.59 4.28 Found 45.23 1.49 3.74

SYNTHESIS EXAMPLE 11 Preparation of2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzamide(11-1) Preparation of2-carboxy-4-[2,3,5,6-tetrafluoro-4-trifluoromethyl-benzyl]-(2,2,2-trifluoroacetyl)amino]phenyltrifluoroacetate

To a solution of2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid (2.00 g, 5.12 mmole) and trifluoroactic anhydride (15 ml) was addedc-H₂SO₄ (0.50 ml) at 10° C. under a nitrogen atmosphere. The reactionmixture was stirred for 20 min at room temperature and then quenchedwith an ice (10 g). After the solvent was removed in vacuo, the residuewas dissolved in ethyl acetate (50 ml). The organic layer was washedwith water (20 ml×2), 10% NaHCO₃ (20 ml×3), 0.5 N HCl (20 ml×2), andwater (20 ml). The organic layer was dried over anhydrous Na₂SO₄. Afterevaporation of the solvent, the residue was recrystallized from ethylacetate/hexane (1:10) to give 1.40 g (47% yield) of2-carboxy-4-[2,3,5,6-tetrafluoro-4-trifluoromethylbenzyl]-(2,2,2-trifluoroacetyl)amino]phenyltrifluoroacetate as an yellow solid.

mp 174-180° C.,

¹H-NMR(DMSO-d₆) δ 5.16(d, 2H), 6.94(d, 1H), 7.44(d, 1H), 7.81(s, 1H),

¹³C-NMR(DMSO-d₆) δ 43.03, 113.2, 114.3, 117.2, 117.8, 118.9, 128.7,130.2, 135.5, 141.8, 143.8, 144.3, 146.2, 146.3, 155.2, 155.5, 161.6,170.5,

IR(neat): 1711, 1673, 1498, 1331, 724 cm⁻¹

(11-2) Preparation of2-carbamoyl-4-[2,3,5,6-tetrafluoro-4-trifluoromethylbenzyl-(2,2,2-trifluororacetyl)amino]phenyltrifluoroacetate

To a solution of2-carboxy-4-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzyl-(2,2,2trifluoroacetyl)amino)phenyl trifluoroacetate (600 mg, 1.05 mmole) inanhydrous methylene chloride (15 ml) was added SOC₂ (1.18 ml, 21.0mmole) at 40° C. under a nitrogen atmosphere. After the reaction mixturewas stirred for 1 hr, the solvent was removed in vacuo. The resultingresidue was dissolved in anhydrous methylene chloride (60 ml) and addedwith ammonium carbonate (assay>30%, 2.00 g). After the reaction mixturewas stirred for 1 hr, it was filtered to remove the remained ammoniumcarbonate. The organic layer was washed with water (40 ml×3) and thendried over anhydrous Na₂SO₄. After evaporation of the solvent, theresidue was recrystallized from methylene chloride/hexane (1:10) to give370 mg (61% yield) of2-carbamoyl-4-[2,3,5,6-tetrafluoro-4-trifluoromethyl-benzyl-(2,2,2-trifluororacetyl)amino]phenyltrifluoroacetate as a white solid.

mp 179-180° C.,

¹H-NMR(DMSO-d₆) δ 5.05(d, 2H), 6.91(d, 1H), 7.20(d, 1H), 7.62(s, 1H),

¹³C NMR(DMSO-d₆) δ 42.31, 114.05, 114.39, 117.25, 118.28, 118.60,127.47, 128.43, 134.14, 141.66, 143.72, 144.20, 146.26, 155.48, 161.19,170.32,

IR(neat) 3415, 3202, 1692, 1673, 1498, 1331 cm⁻¹

(11-3) Preparation of2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzamide

To a solution of2-carbamoyl-4-[2,3,5,6-tetrafluoro-4-trifluoromethylbenzyl-(2,2,2-trifluororacetyl)amino]phenyltrifluoroacetate (300 mg, 0.52 mmole) in methanol (8 ml) and water (3ml) was added c-HCl (2.0 ml) at 40° C. under a nitrogen atmosphere.After the reaction mixture was stirred for 24 hr, the organic solventwas removed in vacuo. The residue was extracted with ethyl acetate (20ml×3). The organic layer was washed with water and then dried overanhydrous Na₂SO₄. After evaporation of the solvent, the residue wasrecrystallized from methylene. chloride/hexane (1:10) to give 120 mg(60% yield) of2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzamideas a white solid.

mp 143-145° C.,

¹H-NMR(DMSO-d₆) δ 4.37(s, 2H), 6.63(d, 1H), 6.76(d, 1H), 7.14(s, 1H),

¹³C NMR(DMSO-d₆) δ 36.25, 111.20, 112.36, 117.44, 121.71, 123.29,123.46, 139.87, 141.61, 143.55, 145.86, 146.07, 153.12, 171.56,

IR(neat) 3453, 3415, 3202, 1692, 1673, 735 cm⁻¹

SYNTHESIS EXAMPLE 12 Preparation of2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzenesulfonic acid

According to the similar procedure in Synthesis Example 1, by using4-amino-2-hydroxybenzene sulfonic acid (1.00 g, 5.30 mmole), DMF (10ml), TEA (1.0 ml), and 2,3,5,6-tetrafluoro-4-trifluoromethylbenzylbromide (0.88 g, 5.30 mmole), 0.61 g (28.0% yield) of2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzenesulfonic acid was obtained as a yellow solid.

mp: above 300° C.,

¹H-NMR(DMSO-d₆) δ 4.28(s, 2H), 5.58(m, 2H), 6.82(s,1H),

¹³C NMR(DMSO-d₆) δ 32.08, 106.41, 111.39, 112.10, 119.15, 119.33,119.51, 126.11, 135.09, 137.17, 139.17, 139.70, 139.89, 140.40, 140.59,141.61,

IR(neat) 3427, 3227, 1492, 1331, 1196, 1135, 628 cm⁻¹

SYNTHESIS EXAMPLE 13 Preparation of methyl2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoate(13-1) Preparation of methyl 5-amino-2-hydroxybenzoate

To a solution of 5-aminosalicylic acid (3.00 g, 19.6 mmole) in methanol(80 ml) was added c-H₂SO₄ (8 ml) at 0° C. After the reaction mixture wasrefluxed for 6 hr, the solvent was removed in vacuo. The resultingresidue was partitioned with ethyl acetate and water. The organic layerwas washed with water (40 ml×3) and then dried over anhydrous MgSO₄.After evaporation of the solvent, the residue was recrystallized fromethyl acetate/hexane to give 2.50 g (76% yield) of methyl5-amino-2-hydroxybenzoate as a yellow solid.

¹H-NMR(CDCl₃) δ 7.2(s, 1H), 6.7(d, 1H), 6.6(d, 1H),

IR(KBr pellet): 3406, 3327, 2950, 1672, 1616, 1492, 1440, 788 cm⁻¹

(13-2) Preparation of methyl2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoate

According to the similar procedure in Synthesis Example 1, by usingmethyl 5-amino-2-hydroxybenzoate (2.00 g, 11.9 mmole), DMF (60 ml), TEA(0.5 ml), and 2,3,5,6-tetrafluoro-4-methylbenzyl bromide (2.60 g, 14.3mmole), 3.20 g (85.0% yield) of methyl2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoatewas obtained as a pale yellow solid:

mp: 127° C.,

¹H-NMR(CDCl₃) δ 7.15(s, 1H), 6.9(d, 1H), 6.7(d, 1H), 4.5(s, 2H), 3.9(s,3H),

IR(KBr pellet): 3382, 1687, 1617, 1586 cm⁻¹ Elemental analysis forC₁₆H₁₀F₇NO₃ % C % H % N % F % O Calculated 48.38 2.54 3.53 33.48 12.08Found 48.12 2.54 3.43

SYNTHESIS EXAMPLE 14 Preparation of2-ethanoyloxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)benzoicacid (14-1) Preparation of 5-tert-butoxycarbonylamino-2-hydroxybenzoicacid

The mixture of 5-aminosalicylic acid (1.01 g, 6.59 mmole), Di-BOC (2.87g, 13.1 mmol), TEA (1.0 ml) in DMF (20.0 ml) was stirred for 2 hr atroom temperature. After the reaction mixture was concentrated, theresidue was dissolved in ethyl acetate. The organic layer was washedwith water and dried over anhydrous Na₂SO₄. After evaporation of thesolvent, the residue was recrystallized from ethyl acetate/hexane togive 1.42 g (84% yield) of 5-tert-butoxy-2-hydroxybenzoic acid as awhite solid.

mp: 282° C.,

¹H-NMR(DMSO-d₆) δ 7.9˜8.0 (s, 1H), 7.4˜7.5 (d, 1H), 6.8˜6.9 (d, 1H),1.4˜1.6 (s, 9H),

¹³C NMR(DMSO-d₆) δ 171.93, 156.50, 152.97, 131.06, 126.36, 119.39,117.03, 113.13, 79.05, 28.42

(14-2) Preparation of 2-ethanoyloxy-5-tert-butoxycarbonylaminobenzoicacid

To a solution of 5-tert-butoxycarbonylamino-2-hydroxybenzoic acid (1.02g, 4.02 mmole) in DMF (20.0 ml) was added with acetyl chloride (39 mg,4.83 mmole) and potassium carbonate (555 mg, 4.02 mmole). The reactionmixture was stirred for 4 hr at room temperature. After the reactionmixture was concentrated, the residue was dissolved in ethyl acetate.The organic layer was washed with water and brine and dried overanhydrous Na₂SO₄. After evaporation of the solvent, the residue wasrecrystallized from ethyl acetate/hexane to give 0.62 g (52% yield) of2-ethanoyloxy-5-tert-butoxycarbonylaminobenzoic acid as a white solid.

mp: 76-78° C.,

¹H-NMR(DMSO-d₆) δ 7.9˜8.0 (s, 1H), 7.4˜7.5 (d, 1H), 6.8˜6.9 (d, 1H),2.0˜2.1 (s, 3H), 1.4˜1.6 (s, 9H),

¹³C NMR(DMSO-d₆) δ 171.88, 165.59, 156.49, 144.71, 131.27, 124.06,79.64, 28.441, 21.13.

(14-3) Preparation of 2-ethanoyloxy-5-aminobenzoic acid

The mixture of 2-ethanoyloxy-5-tert-butoxycarbonylaminobenzoic acid(1.01 g, 3.42 mmole) in TFA/CH₂Cl₂ (20.0 ml. 1:1 v/v) was stirred for 30min at room temperature. After the reaction mixture was concentrated,the residue was dissolved in ether. The resulting residue wasrecrystallized from ethyl acetate/hexane to give 0.65 g (97% yield) of2-ethanoyloxy-5-aminobenzoic acid as a white solid.

mp: 133-136° C.,

¹H-NMR(DMSO-d₆) δ 7.5˜7.6 (s, 1H), 7.2˜7.3 (d, 1H), 7.0˜7.1 (d, 1H),2.1˜2.2 (s, 3H),

¹³C NMR(DMSO-d₆) δ 170.83, 165.57, 143.40, 124.41, 123.45, 121.09,118.64, 113.98, 30.98.

(14-4) Preparation of2-ethanoyloxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid

According to the similar procedure in Synthesis Example 1, by using2-ethanoyloxy-5-aminobenzoic acid (0.81 g, 4.10 mmole) which wasprepared from Synthesis Example (14-3), DMF (15 ml), TEA (0.1 ml),tetrabutyl ammonium iodide (5 mg), and2,3,5,6-tetrafluoro-4-trifluoromethylbenzyl bromide (1.02 ml, 6.15mmole), 0.92 g (53.0% yield) of was obtained2-ethanoyloxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid as a white solid.

mp: 185-187° C.,

¹H-NMR(DMSO-d₆) δ 2.16(s, 3H), 4.46(s, 2H), 6.82(d, 1H), 6.88(d, 1H),7.17(s, 1H),

¹³C NMR(DMSO-d₆) δ 21.64, 36.37, 114.36, 116.97, 123.94, 127.78, 124.81,141.40, 142.44, 144.44, 145.04, 145.84, 146.85, 166.35, 170.18,

IR(KBr pellet): 3410, 1747, 1705, 1489 cm⁻¹ Elemental analysis forC₁₇H₁₀F₇NO₄ % C % H % N % F % O Calculated 48.01 2.37 3.29 31.27 15.05Found 48.05 2.39 3.29

SYNTHESIS EXAMPLE 15 Preparation of2-propanoyloxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid (15-1) Preparation of5-tert-butoxycarbonylamino-2-propanoyloxybenzoic acid

According to the similar procedure in Synthesis Example (14-2), by usingpropanoyl chloride (446 mg, 4.82 mmole), 0.71 g (57.0% yield) of wasobtained 5-tert-butoxycarbonylamino-2-propanoyloxybenzoic acid as awhite solid.

mp: 137-142° C.,

¹H-NMR(DMSO-d₆) δ 7.9˜8.0 (s, 1H), 7.4˜7.5 (d, 1H), 6.8˜6.9 (d, 1H),2.5˜2.6 (t, 2H) 1.4˜1.6 (s, 9H), 1.0˜1.2(q, 3H),

¹³C NMR(DMSO-d₆) δ 172.689, 165.598, 152.804, 144.674, 137.287, 124.045,79.618, 28.358, 27.259, 9.080

(15-2) Preparation of 5-amino-2-propanoyloxybenzoic acid

According to the similar procedure in Synthesis Example (14-3), by using5-tert-butoxycarbonylamino-2-propanoyloxybenzoic acid (1.01 mg, 3.26mmole), 0.67 g (97.0% yield) of was obtained5-amino-2-propanoyloxybenzoic acid as a white solid.

mp: 213-220° C.,

¹H-NMR(DMSO-d₆) δ 7.5˜7.6 (s, 1H), 7.2˜7.3 (d, 1H), 7.0˜7.1 (d, 1H),2.4˜2.6 (t, 2H), 1.0˜1.2 (q, 3H),

¹³C NMR(DMSO-d₆) δ 172.78, 165.35, 145.54, 137.100, 124.90, 124.76,124.23, 121.72, 27.32, 9.10.

(15-3) Preparation of2-propanoyloxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid

According to the similar procedure in Synthesis Example 14-4, by using5-amino-2-propanoyloxybenzoic acid (0.32 g, 1.53 mmole) which wasprepared from Synthesis Example (15-2), DMF (10 ml), TEA (0.05 ml),tetrabutyl ammonium iodide (3 mg), and2,3,5,6-tetrafluoro-4-trifluoromethylbenzyl bromide (0.38 ml, 2.30mmole), 0.34 g (51.0% yield) of was obtained2-propanoyloxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid as a white solid.

mp 188-191° C.,

¹H-NMR(acetone-d₆) δ 1.17 (t, 3H), 2.06 (q, 2H), 4.67 (s, 2H), 6.94 (m,2H), 7.37 (s, 1H),

¹³C NMR(acetone-d₆) δ 8.65, 27.41, 36.34, 114.76, 117.27, 123.57,124.16, 124.59, 141.56, 142.44, 145.29, 145.31, 165.29, 172.77,

IR(KBr pellet): 3414, 1745, 1702, 1491 cm⁻¹ Elemental analysis forC₁₈H₁₈F₇NO₄ % C % H % N % F % O Calculated 49.22 2.75 3.19 30.27 14.57Found 49.20 2.76 3.20

SYNTHESIS EXAMPLE 16 Preparation of2-cyclohexanecarbonyloxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid (16-1) Preparation of5-tert-butoxycarbonylamino-2-cyclohexanecarbonyloxybenzoic acid

According to the similar procedure in Synthesis Example (14-2), by usingcyclohexylcarbonyl chloride (707 mg, 4.82 mmole), 0.72 g (49.0% yield)of was obtained5-tert-butoxycarbonylamino-2-cyclohexanecarbonyloxybenzoic acid as awhite solid.

mp: 68-74° C.,

¹H-NMR(DMSO-d₆) δ 7.9˜8.0 (s, 1H), 7.4˜7.5 (d, 1H), 6.8˜6.9 (d, 1H),2.4˜2.6 (t, 1H), 1.0˜2.0 (m, 19H)

(16-2) Preparation of 5-amino-2-cyclohexanecarbonyloxybenzoic acid

According to the similar procedure in Synthesis Example 14-3, by using5-tert-butoxycarbonylamino-2-cyclohexanecarbonyloxybenzoic acid (1.01mg, 2.78 mmole), 0.70 g (96.0% yield) of was obtained5-amino-2-cyclohexanecarbonyloxybenzoic acid as a white solid.

mp: 116-121° C.,

¹H-NMR(DMSO-d₆) δ 7.5˜7.6 (s, 1H), 7.4˜7.5 (d, 1H), 6.9˜7.0 (d, 1H),2.4˜2.6 (t, 1H), 1.0˜2.0 (m, 10H),

¹³C NMR(DMSO-d₆) 173.87, 170.74, 165.60, 160.09, 158.61, 158.27, 143.14,140,71, 130.03, 124.66, 123.44, 121.70, 119.12, 118.61, 113.81, 42.42,31.25, 28.94, 25.65, 22.38, 14.29.

(16-3) Preparation of2-cyclohexanecarbonyloxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid

According to the similar procedure in Synthesis Example (14-4), by using5-amino-2-cyclohexanecarbonyloxybenzoic acid (1.03 g, 3.95 mmole) whichwas prepared from Synthesis Example (16-2), DMF (15 ml), TEA (0.10 ml),tetrabutyl ammonium iodide (10 mg), and2,3,5,6-tetrafluoro-4-trifluoromethylbenzyl bromide (1.01 ml, 5.92mmole), 0.95 g (49.0% yield) of was obtained2-cyclohexanecarbonyloxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethylbenzylamino)benzoicacid as a white solid.

mp 190-193° C.,

¹H-NMR(acetone-d₆) δ 1.20˜1.57 (m, 6H), 1.64˜1.81 (m, 4H) 2.53 (m, 1H),4.67 (s, 2H), 6.90 (d, 1H), 6.96 (d, 1H), 7.36(S, 1H),

¹³C NMR(acetone-d₆) δ 25.57, 26.07, 36.23, 43.13, 114.67, 117.21,122.21, 122.36, 124.45, 124.56, 142.32, 142.74, 144.21, 145.24, 146.82,165.29, 173.96, IR(KBr pellet): 3402, 1724, 1707, 1491 cm⁻¹ Elementalanalysis for C₂₂H₁₈F₇NO₄ % C % H % N % F % O Calculated 53.56 3.68 2.8426.96 12.97 Found 53.58 3.65 2.85

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 a. The effect of 2-Hydroxy-TTBA on NMDA-induced excitotoxicity.

Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μM NMDA for10 min, alone or with inclusion of 3-300 μM 2-Hydroxy-TTBA. Neuronaldeath was analyzed 24 hr later by measuring levels of LDH released intothe bathing medium, mean±SEM (n=9-12 culture wells per condition),scaled to mean LDH efflux value 24 hr after sham wash (=0) andcontinuous exposure to 500 μM NMDA (=100) that causes near completeneuronal death. *, Significant difference from the vehicle control,p<0.05 using ANOVA and Student-Neuman-Keuls' test.

FIG. 1 b. The effect of 2-Hydroxy-TTS, 2-Hydroxy-TTA, 2-Ethan-TTBA,2-Propan-TTBA, or 2-Cyclohexan-TTBA on NMDA-induced excitotoxicity.

Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μM NMDA for10 min, alone (□) or with inclusion of 3-300 μM 2-Hydroxy-TTS (●),2-Hydroxy-TTA (◯), 2-Ethan-TTBA (▾), 2-Propan-TTBA (∇), or2-Cyclohexan-TTBA (▪). Neuronal death was analyzed 24 hr later bymeasuring levels of LDH released into the bathing medium, mean±SEM(n=9-12 culture wells per condition), scaled to mean LDH efflux value 24hr after sham wash (=0) and continuous exposure to 500 μM NMDA (=100).*, Significant difference from the vehicle control, p<0.05 using ANOVAand Student-Neuman-Keuls' test.

FIG. 2 a. Blockade of NMDA currents by 2-Hydroxy-TTBA.

Typical NMDA-induced inward currents (NMDA currents) were evoked byapplying 300 μM NMDA to cortical neurons which were held at −60 mV.Successive application of various concentrations of 2-Hydroxy-TTBAreduced the response elicited by 300 μM NMDA in aconcentration-dependent manner (n=8). The graph shows a dose responserelation of 2-Hydroxy-TTBA to NMDA currents; its IC₅₀ value was close to35 μM and Hill coefficient 0.91.

FIG. 2 b. Pretreatment with 2-Hydroxy-TTBA did not influence NMDAcurrents.

Cortical neurons were treated with 100 μM 2-Hydroxy-TTBA, washedthoroughly, and then applied with 300 μM NMDA. The pretreatment with2-Hydroxy-TTBA did not influence NMDA-induced inward currents,suggesting that 2-Hydroxy-TTBA exert its blocking action only when thereceptor has been activated by agonist (n=6).

FIG. 3 a. The effect of 2-Hydroxy-TTBA on FeCl₂-induced oxidativestress.

Mouse cortical cell cultures (DIV 12-14) were exposed to continuously to50 μM Fe²⁺, alone or with inclusion of 0.1-100 μM 2-Hydroxy-TTBA (●) ortrolox (◯), a membrane-permeable form of vitamin E). Neuronal death wasanalyzed 24 hr later by measuring levels of LDH released into thebathing medium, mean±SEM (n=9-12 culture wells per condition), scaled tomean LDH efflux value 24 hr after sham wash (=0) and continuous exposureto 500 μM NMDA (=100). *, Significant difference from the vehiclecontrol, p<0.05 using ANOVA and Student-Neuman-Keuls' test.

FIG. 3 b. The effect of 2-Hydroxy-TTS, 2-Hydroxy-TTA, 2-Chloro-TTP,2-Ethan-TTBA, 2-Propan-TTBA, or 2-Cyclohexan-TTBA on FeCl₂-inducedoxidative stress.

Mouse cortical cell cultures (DIV 12-14) were exposed to continuously to50 μM Fe²⁺, alone (♦) or with inclusion of 1-30 μM 2-Hydroxy-TTS (●),2-Hydroxy-TTA (◯), 2-Chloro-TTP (▾), 2-Ethan-TTBA (∇), 2-Propan-TTBA(▪), or 2-Cyclohexan-TTBA (□). Neuronal death was analyzed 24 hr laterby measuring levels of LDH released into the bathing medium, mean±SEM(n=9-12 culture wells per condition), scaled to mean LDH efflux value 24hr after sham wash (=0) and continuous exposure to 500 μM NMDA (=100).*, Significant difference from the vehicle control, p<0.05 using ANOVAand Student-Neuman-Keuls' test.

FIG. 4. The effect of 2-Hydroxy-TTBA on SNP-induced oxidative stress.

Mouse cortical cell cultures (DIV 12-14) were exposed to continuously 5μM sodium nitroprusside (SNP), alone (●) or with inclusion of 0.1-10 mMaspirin (◯), 1-100 μM trolox (▾), or 0.1-10 μM 2-Hydroxy-TTBA(∇).Neuronal death was analyzed 24 hr later by measuring levels of LDHreleased into the bathing medium, mean±SEM (n=9-12 culture wells percondition), scaled to mean LDH efflux value 24 hr after sham wash (=0)and continuous exposure to 500 μM NMDA (=100). *, Significant differencefrom the vehicle control, p<0.05 using ANOVA and Student-Neuman-Keuls'test.

FIG. 5. The effect of 2-Hydroxy-TTBA on zinc toxicity.

Mouse cortical cell cultures (DIV 12-14) were exposed to 300 μM Zn²⁺ for30 min, alone or with inclusion of 3-300 μm 2-Hydroxy-TTBA. Neuronaldeath was analyzed 24 hr later by measuring levels of LDH released intothe bathing medium, mean±SEM (n=9-12 culture wells per condition),scaled to mean LDH efflux value 24 hr after sham wash (=0) andcontinuous exposure to 500 μM NMDA (=100).

FIG. 6 a. Free radical scavenging activity of 2-Hydroxy-TTBA.

2-Hydroxy-TTBA (●) or trolox (◯) was reacted with 100 uM1,1-diphenyl-2-picrylhydrazil (DPPH, a stable free radical) dissolved inethanol for 10 min. The radical scavenging activity was determined bymeasuring the decrease in DPPH levels at 517 nm. *, Significantdifference from the vehicle control, p<0.05 using ANOVA andStudent-Neuman-Keuls' test.

FIG. 6 b. The DPPH assay of 2-Hydroxy-TTS, 2-Hydroxy-TTA, or2-Chloro-TTP.

2-Hydroxy-TTS (●), 2-Hydroxy-TTA (◯), 2-Chloro-TTP (▾) or control (∇)was reacted with 100 uM DPPH dissolved in ethanol. The radicalscavenging activity was determined by measuring the decrease in DPPHlevels at 517 nm. *, Significant difference from the vehicle control,p<0.05 using ANOVA and Student-Neuman-Keuls' test.

FIG. 7 a. 2-Hydroxy-TTBA reduces neuronal death in the spinal cord in ananimal model of ALS.

Bright field photomicrographs of spinal cord sections stained with eosinfrom amyotrophic lateral sclerosis (ALS) mice over expressing the mutantSOD1-G93A that received vehicle (A) or 2-Hydroxy-TTBA (B, 10 mg/kg/daythrough drinking water) for 8 weeks from the age of 2 months.

FIG. 7 b. 2-Hydroxy-TTBA reduces neuronal death in the spinal cord in ananimal model of ALS.

Degenerating neurons from FIG. 7 a. were analyzed by counting viableneurons after staining with eosin in the dorsal and ventral horn ofspinal cord from ALS mice treated with a vehicle (Control) or2-Hydroxy-TTBA, mean±S.E.M (n=5 animals for each condition). *,Significant difference from the vehicle control, p<0.05 using theindependent t-test.

FIG. 8 a. Intraperitoneal administration of 2-Hydroxy-TTBA reducesischemic injury in brain.

Adult rats received transient cerebral ischemia by occluding rightmiddle cerebral artery and both common carotid arteries for 60 min withintraperitoneal injections of vehicle or 50 mg/kg 2-Hydroxy-TTBA at 5min, 30 min, or 1 hr after reperfusion. Infarct volume was analyzed 24hr later after staining brain slices with 2% 2,3,5-triphenyltetrazoliumchloride (TTC), mean±SEM (n=8-11 rats per each condition). *,Significant difference from the vehicle control, p<0.05 using ANOVA andStudent-Neuman-Keuls' test.

FIG. 8 b. Intraperitoneal administration of 2-Hydroxy-TTBA reducesischemic injury in brain.

Adult rats received transient cerebral ischemia by occluding rightmiddle cerebral artery and both common carotid arteries for 60 min withintraperitoneal injections of vehicle (control) or 50 or 100 mg/kg2-Hydroxy-TTBA at 5 min after reperfusion. Infarct volume was analyzed24 hr later after staining brain slices with 2%2,3,5-triphenyltetrazolium chloride (TTC), mean±SEM (n=7-8 rats per eachcondition). *, Significant difference from the vehicle control, p<0.05using ANOVA and Student-Neuman-Keuls' test.

FIG. 8 c. Intravenous injections of 2-Hydroxy-TTBA reduces ischemicinjury in brain.

Adult rats received transient cerebral ischemia by occluding rightmiddle cerebral artery and both common carotid arteries for 60 min, withintravenous injections of vehicle (control) or 5 mg/kg 2-Hydroxy-TTBA at30 min after occlusion or 5 min, 30 min, 1 hr, 2 hr, or 4 hr afterreperfusion. Infarct volume was analyzed 24 hr later, mean±SEM (n=8-11rats per each condition). *, Significant difference from the vehiclecontrol, p<0.05 using ANOVA and Student-Neuman-Keuls' test.

FIG. 8 d. Intravenous injections of 2-Hydroxy-TTBA reduces ischemicinjury in brain.

Adult rats received transient cerebral ischemia by occluding rightmiddle cerebral artery and both common carotid arteries for 60 min, withintravenous injections of vehicle (control) or 1, 2.5, 5, 10 or 20 mg/kg2-Hydroxy-TTBA at 5 min after reperfusion. Infarct volume was analyzed24 hr later, mean±SEM (n=8-10 rats per each condition). *, Significantdifference from the vehicle control, p<0.05 using ANOVA andStudent-Neuman-Keuls' test.

FIG. 8 e. Oral administration of 2-Hydroxy-TTBA reduces ischemic injuryin brain.

Adult rats received transient cerebral ischemia by occluding rightmiddle cerebral artery and both common carotid arteries for 60 min, withoral administrations of vehicle (control) or 10 or 20 mg/kg2-Hydroxy-TTBA at 5 min after reperfusion. Infarct volume was analyzed24 hr later, mean±SEM (n=8-10 rats per each condition). *, Significantdifference from the vehicle control, p<0.05 using ANOVA andStudent-Neuman-Keuls' test.

FIG. 9 a. Effects of 2-Hydroxy-TTBA on mitochondrial ROS generation at72 hr of recirculation after global ischemia.

Mitotracker CM-H₂X ROS was injected into the lateral ventricle of ratbrain at 24 hr before ischemic surgery, and then animals receivedtransient forebrain ischemia for 10 min, with intraperitoneal injectionsof vehicle (control) or 2-Hydroxy-TTBA (50 mg/kg) at 5 min afterreperfusion. The levels of mitochondrial ROS were analyzed 2 hr later,mean±S.E.M. (n=50-70 neurons randomly chosen from the CA1 sector of thehippocampal formation per each condition). *, Significant differencefrom the vehicle control, p<0.05 using the independent t-test.

FIG. 9 b. 2-Hydroxy-TTBA prevents neuronal death in the CA1 sector at 72hr of recirculation after global ischemia.

Adult rats received transient global ischemia for 10 min, withintraperitoneal injections of vehicle (control) or 2-Hydroxy-TTBA (50mg/kg) at 5 min after reperfusion. The number of degenerating neurons inthe CA1 was analyzed 3 d later by counting viable neurons after stainingwith cresyl violet, mean±S.E.M. *, Significant difference from thevehicle control, p<0.05 using the independent t-test.

FIG. 10 a. 2-Hydroxy-TTBA reduces death of dopaminergic neurons in thesubstantia nigra 3 d following MPTP injection.

Bright field photomicrographs showing dopaminergic neurons in thesubstantia nigra immunostained with anti-tyrosine hydroxylase (TH)antibody 3 d following sham control (A) or the single daily injection of1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP, 40 mg/kg, SC) inC57/BL6 mice, alone (B) or with intraperitoneal injections (2 times perday) of 2-Hydroxy-TTBA (C, 25 mg/kg; D, 50 mg/kg) prior to MPTPadministration.

FIG. 10 b. 2-Hydroxy-TTBA reduces death of dopaminergic neurons in thesubstantia nigra 3 d following MPTP injection.

Quantification of TH-positive dopaminergic neurons in the substantianigral sections 3 d following the single daily injection of (MPTP, 40mg/kg, SC), alone or with pretreatment with 2-Hydroxy-TTBA as describedabove. *, Significant difference from the control, p<0.05 using ANOVAand Student-Neuman-Keuls' test.

FIG. 10 c. 2-Hydroxy-TTBA reduces death of dopaminergic neurons in thesubstantia nigra 7 d following MPTP injection.

Bright field photomicrographs showing dopaminergic neurons in thesubstantia nigra immunostained with anti-tyrosine hydroxylase (TH)antibody 7 d following sham control (A) or the single daily injection of1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP, 40 mg/kg, SC) inC57/BL6 mice, alone (B) or with intraperitoneal injections (2 times perday) of 2-Hydroxy-TTBA (C, 25 mg/kg; D, 50 mg/kg) prior to MPTPadministration.

FIG. 11. 2-Hydroxy-TTBA prevents generation of mitochondrial ROS in thedorsal horn neurons of the spinal cord following traumatic injury.

Adult rats received compression injury at the level of T8 spinal cord,alone (control) or with injections of 2-Hydroxy-TTBA (50 mg/kg, ip) andMitotracker CM-H₂X ROS immediately after injury. Animals were euthanized2 d later, spinal cord sections sections immunostained with an antibodyfor NeuN (a neuronal marker protein), and levels of mitochondrial ROS inthe dorsal horn neurons (DHN) analyzed by measuring fluorescenceintensity of oxidized Mitotracker CM-H₂X, mean±S.E.M. [n=12 rats foreach condition (5 spinal cord sections for each rat)]. Significantdifference from the vehicle control, p<0.05 using the independentt-test.

This present invention is described particularly in experimentalexamples using 2-Hydroxy-TTBA, 2-Hydroxy-TTS, 2-Hydroxy-TTA,2-Chloro-TTP, 2-Ethan-TTBA, 2-Propan-TTBA and 2-Cyclohexan-TTBAmanufactured in synthesis example as follows.

However, the examples described below are just representative of thepresent invention, which could include more examples.

EXAMPLE 1 mixed cortical cell cultures of neurons and glia

For mixed neuron-glia culture, mouse cerebral cortices were removed frombrains of the 14-16 day-old-fetal mice (E14-16), gently triturated andplated on 24 well plates (2×10⁵ cells/plate) precoated with 100 μg/mlpoly-D-lysine and 4 μg/ml laminine. Cultures were maintained at 37° C.in a humidified 5% CO₂ atmosphere. Plating media consist of Eaglesminimal essential media (MEM, Earles salts, supplied glutamine-free)supplemented with 5% horse serum, 5% fetal bovine serum, 26.5 mMbicarbonate, 2 mM glutamine, and 21 mM glucose.

After 7-8 days in vitro (DIV 7-8), 10 μM cytosine arabinofuranoside(Ara-C) was included to halt overgrowth of glia. The drug treatment wascarried on DIV 12-15 cortical cell culture. Overall neuronal cell injurywas assessed by measuring amount of lactate dehydrogenase (LDH) releasedinto the bathing medium 24 hr after neurotoxic insults as previouslydescribed [Koh and Choi, J Neurosci Methods 20:83-90, 1987].

EXAMPLE 2 Inhibitory effects of excitotoxicity by 2-hydroxy-TTBA,2-hydroxy-TTA, 2-Hydroxy-TTS, 2-Ethan-TTBA, 2-Prppan-TTBA or2-Cyclohexan-TTBA

At DIV 13-15 in mixed neuron-glia culture as shown in Example 1,cortical cell cultures were exposed to 300 μM NMDA for 10 min, alone orwith 3-300 μM 2-Hydroxy-TTBA, 10-300 μm 2-Hydroxy-TTS, 30-300 μM2-Hydroxy-TTA, 30-300 μM 2-Ethan-TTBA, 10-300 μM 2-Prppan-TTBA, or10-300 μM 2-Cyclohexan-TTBA. Neuronal death was assessed 24 hr later bymeasuring amount of LDH released into the bathing medium. *, Significantdifference from the vehicle control, p<0.05 using ANOVA andStudent-Neuman-Keuls' test.

Cortical cell cultures exposed to 300 μM NMDA for 10 min underwentwidespread neuronal death (approximately 75-80% neurons died) over thenext 24 hr. NMDA-induced neuronal death was blocked by co-treatment with2-Hydroxy-TTBA in a dose-dependent manner at doses of 10-300 μM (FIG. 1a).

Concurrent administration of 2-Hydroxy-TTS, 2-Hydroxy-TTA, 2-Ethan-TTBA,2-Propan-TTBA, or 2-Cyclohexan-TTBA also prevented NMDA-induced neuronaldeath at doses of 30-300 μM. (FIG. 1 b).

EXAMPLE 3 Blocking effect of 2-Hydroxy-TTBA on NMDA-induced inwardcurrents

Whole cell recordings were performed on cortical cell cultures at roomtemperature as described [Seo et al., J. Pharmacol. Exp. Ther.,299:377-384(2001)]. Typical NMDA-induced inward currents (NMDA currents)were evoked immediately after applying 300 μM NMDA to cultured corticalneurons which were held at −70 mV. Bath application of 2-Hydroxy-TTBAimmediately depressed NMDA-evoked currents in a dose dependent manner(n=7-15 neurons/condition, IC₅₀ value=30.55±2.96 μM) (FIG. 2 a).Application of 100 μM 2-Hydroxy-TTBA alone had no effect on the holdingcurrents and had little effect on the response of the cell to asubsequent treatment of 300 μM NMDA (FIG. 2 b), suggesting that2-Hydroxy-TTBA exerts antagonistic effect only when the NMDA receptor isactivated (n=6).

EXAMPLE 4 Blockade of oxidative neuronal death by 2-Hydroxy-TTBA,2-Hydroxy-TTA, 2-Hydroxy-TTS, 2-Chloro-TTP, 2-Ethan-TTBA, 2-Propan-TTBAor 2-Cyclohexan-TTBA (4-1) Inhibition of FeCl₂-Induced Free RadicalToxicity

Mixed cortical cell cultures (DIV 13-15) were continuously exposed to 50μM FeCl₂, which produces hydroxyl radical via a Fenton reaction, aloneor with inclusion of 2-Hydroxy-TTBA, 2-Hydroxy-TTA, 2-Hydroxy-TTS,2-Chloro-TTP, 2-Ethan-TTBA, 2-Propan-TTBA, 2-Cyclohexan-TTBA or trolox(a vitamin E analogue) at indicated doses. Neuronal cell death wasanalyzed 24 hr later by LDH assay as described above. *, Significantdifference from the vehicle control (FeCl₂), p<0.05 using ANOVA andStudent-Neuman-Keuls' test.

Cortical cell cultures exposed to FeCl₂ underwent widespread neuronaldeath over the next 24 hr. 2-Hydroxy-TTBA and trolox preventedFeCl₂-induced free radical neurotoxicity in a dose-dependent manner.However, 2-Hydroxy-TTBA was 30-fold stronger than trolox in preventingfree radical neurotoxicity (FIG. 3 a). Moreover, synthetic derivativesof 2-Hydroxy-TTBA (2-Hydroxy-TTS, 2-Hydroxy-TTA, 2-Chloro-TTP,2-Ethan-TTBA, 2-Propan-TTBA, or 2-Cyclohexan-TTBA) showed much strongerneuroprotective effects than trolox in preventing FeCl₂-induced freeradical neurotoxicity (FIG. 3 b).

(4-2) Inhibition of SNP (sodium nitroprusside) cytotoxicity

Mixed cortical cell cultures were continuously exposed to 5 μM SNP, anitric oxide (NO) donor, alone or with inclusion of 0.1-10 μM2-Hydroxy-TTBA, 1-10 μM trolox or 100-10,000 μM aspirin. Neuronal celldeath was analyzed 24 hr later by LDH assay. Significant difference fromthe vehicle control (SNP), p<0.05 using ANOVA and Student-Neuman-Keuls'test.

Administration of SNP resulted in complete neuronal cell death and someglial cell death. SNP toxicity was completely blocked in the presence of2-Hydroxy-TTBA and trolox. The former was 100-fold stronger than thelatter in preventing NO toxicity. Aspirin, a structural component of2-Hydroxy-TTBA, did not reduce SNP cytotoxicity (FIG. 4).

(4-3) Inhibition of Zinc Neurotoxicity

To induce zinc (Zn²+) neurotoxicity, mixed cortical cell cultures wereexposed to 100 μM ZnCl₂ for 30 min in a HEPES-buffered control saltsolution (HCSS): (in mM) 120 NaCl, 5 KCl, 1.6 MgCl₂, 2.3 CaCl₂, 15glucose, 20 HEPES and 10 NaOH, alone or with 3-300 uM 2-Hydroxy-TTBA.After exposure, cultures were washed out 3 times and exchanged with MEMadjusted to 25 mM glucose and 26.2 mM sodium bicarbonate. Neuronal celldeath was analyzed 24 hr later by LDH assay. *, Significant differencefrom the vehicle control (SNP), p<0.05 using ANOVA andStudent-Neuman-Keuls' test.

Concurrent treatment with 2-Hydroxy-TTBA prevented zinc-induced neuronaldeath in a dose-dependent manner at doses of 10-300 μM (FIG. 5).

(4-4) DPPH assay of 2-Hydroxy-TTBA, 2-Hydroxy-TTA, 2-Hydroxy-TTS or2-Chloro-TTP

To examine the free radical scavenging effects, 1-100 μM 2-Hydroxy-TTBA,2-Hydroxy-TTA, 2-Hydroxy-TTS, 2-Chloro-TTP or trolox was reacted with100 μM DPPH (2,2-diphenyl-1-picryl-hydrazyl radical), a stable freeradical, dissolved in ethanol. After incubation for 30 min, relativedecrease in DPPH absorption at 517 nm was measured by aspectrophotometer, mean±SEM (n=3 test tubes per condition). *,Significant difference from the vehicle control (DPPH alone), p<0.05using ANOVA and Student-Neuman-Keuls' test.

Compared to the anti-oxidant trolox, 2-Hydroxy-TTBA reduced levels ofDPPH at lower doses, suggesting that 2-Hydroxy-TTBA is a directanti-oxidant stronger than trolox (FIG. 6 a). Other syntheticderivatives also reduced levels of DPPH (FIG. 6 b).

EXAMPLE 5 Prevention of neuronal cell death in spinal cord of ALS mouseby 2-hydroxy-TTBA

Transgenic mice with the G93A human SOD1 mutation(B6SJL-TgN(SOD1-G93A)1Gur), animal models of ALS (ALS mice), wereobtained from Jackson Laboratories (Me., USA). 2-hydroxy-TTBA wasadministered to 2 month-old wild type and ALS transgenic mice throughdrinking water bottle (10 mg/kg per day) for 8 weeks. Animals were theneuthanized and spinal cords processed for histological examination bystaining with hematoxylin and eosin. Spinal motor neurons from ALS micewithout being treated with 2-hydroxy-TTBA (control) underwentdegeneration as evident by eosinophilic staining, which was reduced inALS mice treated with 2-hydroxy-TTBA (FIG. 7 a).

The number of degenerating neurons was analyzed by counting eosinophilicneurons in ventral horn (VH) and dorsal horn (DH). Administration of2-hydroxy-TTBA significantly prevented degeneration of dorsal andventral horn neurons in the spinal cord from ALS mice (FIG. 7 b). *,Significant difference from the control, p<0.05 using independence ttest.

EXAMPLE 6 Prevention of hypoxic-ischemic brain injury by 2-Hydroxy-TTBA(6-1) Induction of transient focal cerebral ischemia in rat

Sprague-Dawley rats were anesthetized with chloral hydrate, andsubjected to focal cerebral ischemia by occlusion of middle cerebralartery (MCAO) as previously described [Tamura et al., J. Cerebr. BloodFlow Metab. 1:53-60(1981)]. Both common carotid arteries (CCAS) wereexposed and right middle cerebral artery (rMCA) was exposed under thesurgical microscope by making a 3 mm diameter craniotomy rostral to theforamen ovale. CCAs and rMCA were occluded with microclips. The clipswere released 60 min later and restoration of blood flow in rMCA wasobserved under the microscope

(6-2) 2-Hydroxy-TTBA reduces infarct volume after 60 min MCAO

Animals received MCAO for 60 min, alone or with administration of2-Hydroxy-TTBA (50 mg/kg, i.p.) at indicated points of time afterreperfusion. Saline was injected as a control. Animals were euthanized24 hr later and brains removed and sectioned coronally into seven 2-mmslices in a brain matrix. Brain slices were placed in 2%2,3,5,-triphenyltetrazolium chloride solution, followed by 10% formalinovernight. The infarction area, outline in white, was measured (TINAimage analysis system) and infarction volume was calculated by summingthe infarct volume showing white of sequential 2-mm-thick section,mean±SEM (n=8-12 rats/condition) (FIG. 8 a). Note that delayedadministration (ip) of 2-Hydroxy-TTBA up to 1 hr after reperfusionsignificantly reduced infarct volume. Higher doses of 2-Hydroxy-TTBA(100 mg/kg, ip) also showed similar protection against 60 min MCAO (FIG.8 b).

Additional experiments were performed to examine effects of intravenousinjections of 2-Hydroxy-TTBA (5 mg/kg) against 60 min MCAO.2-Hydroxy-TTBA was administered at indicated points of time afterreperfusion. The delayed injections of 2-Hydroxy-TTBA up to 4 hr afterreperfusion significantly attenuated infarct volume evolving 24 hr after60 min MCAO, mean±SEM (n=8-12 ratS/condition) (FIG. 8 c).

Dose-response experiments of the intravenous injections of2-Hydroxy-TTBA showed that 2-Hydroxy-TTBA attenuated infarct volume atdoses as low as 1 mg/kg. The protective effects of 2-Hydroxy-TTBA wereobserved at doses higher than 2.5 mg/kg, mean±SEM (n=8-12rats/condition) (FIG. 8 d).

Protective effects of 2-Hydroxy-TTBA were also verified through oraladministration. In particular, administration of 2-Hydroxy-TTBA (10 or20 mg/kg, p.o.) at 5 min after reperfusion reduced infarct volume 24 hrafter 60 min MCAO, mean±SEM (n=8-12 ratS/condition) (FIG. 8 e).

EXAMPLE 7 Prevention of the CA1 neuronal cell death after transientforebrain ischemia by 2-Hydroxy-TTBA (7-1) Induction of Global Ischemiain Rat

Male adult Sprague-dawley rats (250-300 g) were anesthetized withchloral hydrate and subjected to four-vessel occlusion model (occlusionof both common carotid arteries and vertebral arteries) as previouslydescribed (Pulsinelli and Brierley, stroke 10: 267-272 (1979)].

(7-2) Inhibition of mitochondrial ROS generation after global ischemiaby 2-Hydroxy-TTBA

Rats received the intracerebroventricular injections of 0.4 nmolMitotracker CM-H₂X Ros. After 24 hr, rats received four-vessel occlusionfor 10 min. Immediately after reperfusion, rats received theintraperitoneal injections of saline (control) or 2-Hydroxy-TTBA (50mg/kg). Levels of mitochondrial ROS were analyzed 2 hr later bymeasuring the fluorescence intensity of oxidized Mitotracker CM-H₂X Rosin mitochondria. Administration of 2-Hydroxy-TTBA reduced production ofmitochondrial ROS 2 hr after transient global ischemia (FIG. 9 a).

(7-3) Inhibition of neuronal cell death in the CA1 field followingtransient forebrain ischemia by 2-Hydroxy-TTBA

Rats received four-vessel occlusion for 10 min followed byadministration of saline (control) or 2-Hydroxy-TTBA (50 mg/kg, ip).Animals were euthanized 3 d later and processed for analysis of neuronaldeath in the CA1 field after staining with hematoxylin and eosin(mean±SEM, N=12 rats/condition). Administration of 2-Hydroxy-TTBAprevented delayed neuronal cell death in the CA1 areas evolving aftertransient global ischemia (FIG. 9 b). *, Significant difference from thecontrol, p<0.05 using independence t test.

EXAMPLE 8 Prevention of dopaminergic neuronal cell death in thesubstantial nigra following the injection of MPTP(1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine) by 2-Hydroxy-TTBA (8-1)Inhibition of dopaminergic neurons by 2-Hydroxy-TTBA

C57/BL6 mice (male, 8 weeks) received the injections of 40 mg/kg MPTP(s.c.), alone or with 25 or 50 mg/kg 2-Hydroxy-TTBA (ip) every 12 hr perday beginning 30 min before MPTP injection. After 3 or 7 days, allanimals were anesthetized with chloral hydrate and perfusedtranscardially with PBS followed by 4% paraformaldehyde. Brains wereimmediately removed and immersed in the fixative for 8-10 h. Thefixative was replaced with 30% sucrose, incubated at 4° C. for 2 d, andstored at −70° C. The brains were sectioned at a thickness of 30 μm on asliding microtome (TPI, Inc., MO). Sections were then stored in 0.1 Mphosphate buffer (pH 7.4, 30% (v/v) glycerol, 30% ethylene glycol) at 4°C. until use. The sections were immunostained with anti-TH (tyrosinehydroxylase) antibody, colored by DAB (diaminobenzidine), and thenobserved under light microscope to analyze the degeneration ofdopaminergic neurons.

Mice treated with MPTP showed marked degeneration of the dopaminergicneurons in the substantia nigra over the next 3 d. The intraperitonealinjections of 2-Hydroxy-TTBA significantly attenuated degeneration ofthe dopaminergic neurons (FIGS. 10 a and 10 b). The protective effectsof 2-Hydroxy-TTBA are also observed at 7 d following administration ofMPTP (FIGS. 10 c and 10 d).

EXAMPLE 9 Inhibition of Mitochondrial ROS Production in the Spinal Cordafter Traumatic Spinal Cord Injury

Sprague-Dawley rats (250-300 g, female) received traumatic spinal cordinjury by compression of the dorsal spinal cord. T8 segment of spinalcord was exposed on the dorsal side, compressed with 20 g for 10 min,and then mitochondrial CM-H₂X Ros was immediately injected into thespinal cord. 2-Hydroxy-TTBA (50 mg/kg, ip) or a vehicle control(control) was injected with mitochondrial CM-H₂X Ros. Levels ofmitochondrial ROS in the dorsal horn neurons were analyzed 48 hr lateras described above after immunolabeling with anti-NeuN antibody, aneuron-specific marker. Administration of 2-Hydroxy-TTBA significantlyreduced mitochondrial ROS production in the spinal cord neurons afterthe traumatic spinal cord injury (FIG. 11)(mean±SEM, N=12rats/condition). *, Significant difference from the control, p<0.05using independence t test.

As described above, tetrafluorobenzyl derivatives or itspharmaceutically-acceptable salts in the present invention can be usedas NMDA receptor antagonists, anti-oxidants, and inhibitors of zincneurotoxicity.

The concrete diseases applicable with tetrafluorobenzyl derivatives orits pharmaceutically-acceptable salts are described as follows.

Application examples described below are part of examples of thisinvention. This invention is not limited to application examples.

APPLICATION EXAMPLE 1 Stroke

Interrupted blood supply to brain or stroke induces neuronal deathprimarily through over-activation of Ca²⁺-permeable glutamate receptorinduced by accumulation of glutamate, an excitatory neurotransmitter, insynaptic cleft. It has been well documented that NMDA receptorantagonists decrease the neuronal cell death by ischemic strokeaccounting for 80% of stroke [Simon et al., Science, 226:850-852 (1984);Park et al., Ann Neurol., 24:543-551 (1988); Wieloch, Science,230:681-683 (1985); Kass et al.; Exp. Neurol., 103:116-122 (1989); Weisset al., Brain Res., 380:186-190 (1986)]. It has also been reported thatROS and zinc are main mechanism of neuronal death following stroke.Anti-oxidants or inhibitors of zinc toxicity protect ischemic injury inanimal models of stroke (Flamm, E. S. et al., Stroke,9(5):445-447(1978); Kogure, K. et al., Prog. Brain Res.,63:237-259(1985); Chan, P. H., J. Neurotrauma., 9suppl2:S417-423(1992)]. Therefore, the compounds in the presentinvention showing multiple protective effects against excitotoxicity,oxidative stress, and zinc toxicity can be used as therapeutic drugs forstroke.

APPLICATION EXAMPLE 2 Trauma

Excitotoxins are closely related to the degeneration of neuronal cellsfollowing traumatic brain injury (TBI) and traumatic spinal cord injury(TSCI). quinolinic acid, an NMDA receptor agonist present in human body,is increased 50 to 500 times in TBI patients [Sinz et al., J. Cereb.Blood Flow Metab., 18:610-615 (1988)]. It has been reported that NMDAreceptor antagonists decrease the neuronal death following TBI and TSCI[Faden et al., J Neurotrauma, 5:33-45 (1988); Okiyama et al., JNeurotrauma, 14:211-222 (1997)]. Anti-oxidants also inhibit tissuedamage following TBI or TSCI [Faden & Salzman, Trends Pharmacol. Sci.,13:29-35(1992)]. Therefore, the compounds in the present inventionshowing multiple protective effects against excitotoxicity and oxidativestress can be used as therapeutic drugs for TBI and TSCI.

APPLICATION EXAMPLE 3 Epilepsy

Administration of kainate, an agonist of AMPA and kainate glutamatereceptors, induces seizure and neuronal cell death in several brainareas including the hippocampal formation. NMDA receptor antagonistswere shown to inhibit convulsion and seizure in several epileptogenicanimal models [Anderson et al., J. Neurophysiol, 57:1-21 (1987); Wong etal., Neurosci Lett., 85:261-266 (1988); Mc Namara et al.,Neuropharmacology, 27:563-568 (1988)]. Anti-oxidants inhibit seizure andseizure-induced neuronal death [He et al., Free Radic. Biol. Med.22:917-922(1997); Kabuto et al., Epilepsia, 39:237-243(1998)].Therefore, the compounds in the present invention showing multipleprotective effects against excitotoxicity and oxidative stress can beused as therapeutic drugs for epilepsy and seizure-induced neuronaldeath.

APPLICATION EXAMPLE 4 Amyotrophic Lateral Sclerosis

ALS patients show increased levels of extracellular glutamate anddefects in glutamate transport in astrocytes. Administration ofglutamate receptor agonists into the spinal cord mimicked pathologicalchanges in the spinal cord of ALS patients [Rothstein et al., ClinNeurosci., 3:348-359 (1995); Ikonomidou et al., J Neuropathol ExpNeurol, 55:211-224 (1996)]. Besides excitotoxicity, evidence is beingaccumulated that oxidative stress is involved in neuronal death in ALS[Cookson & Shaw, Brain Pathol., 9:165-186(1999)]. In fact, the majorpharmacological action of riluzole, the new drug for ALS patients thatreceived FDA approval, involves prevention of excitotoxicity andoxidative stress [Obrenovitch, Trends. Pharmacol. Sci. 19:9-11(1998);Noh et al., Neurobiol. Dis., 7:375-383(2000)]. Therefore, the compoundsin the present invention showing multiple protective effects againstexcitotoxicity and oxidative stress can be used as therapeutic drugs forALS.

APPLICATION EXAMPLE 5 Parkinson's Disease (PD)

PD is a neurodegenerative disease showing the disorder of motor functionby a selective death of dopaminergic neurons in the substantia nigra.Several antagonists of NMDA receptors protect dopaminergic neurons fromthe dopaminergic neurotoxin MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) [Lange et al., NaunynSchmiedebergs Arch.Pharmacol. 348:586-592 (1993); Brouillet and Beal.Neuroreport. 4:387-390 (1993)]. NMDA receptor antagonists alsoameliorate levodopa-induced dyskinesia and thus can improve thetherapeutic effects of levodopa [Papa and Chase, Ann.Neurol. 39:574-578(1996); Marin et al., Brain Res. 736:202-205 (1996)]. Oxidative stressas well as excitotoxicity has been proved as a main mechanism ofneuronal cell death in PD patients [Schapira et al., biochem. Soc.Trans., 21:367-370(1993)]. Therefore, the compounds in the presentinvention showing multiple protective effects against excitotoxicity andoxidative stress can be used as therapeutic drug for PD.

APPLICATION EXAMPLE 6 Huntington's Disease (HD)

HD is a progressive neurodegenerative disease predominantly affectingsmall- and medium-sized interneurons in the striata. These pathologicalfeatures of HD are observed in vivo and in vitro followingadministration of NMDA receptor agonists, raising the possibility thatNMDA receptor-mediated neurotoxicity contributes to selective neuronaldeath in HD [Koh et al., Science 234:73-76 (1986); Beal et al., Nature321:168-171 (1986); Beal et al., J. Neurosci. 11:1649-1659 (1991)].Since evidence is being accumulated that oxidative stress, such asmitochondrial dysfunction and generation of ROS, causes neuronal deathobserved in PD, it is possible that the drugs inhibiting ROS are usedfor therapy of HD [Jenner, Pathol. Biol. 44:57-64(1996); Albers & Beal,J. Neural. Transm. Suppl., 59:133-154(2000)]. Therefore, the compoundsin the present invention showing multiple protective effects againstexcitotoxicity and oxidative stress can be used as therapeutic drugs forHD.

APPLICATION EXAMPLE 7 Alzheimer's Disease (AD)

The degeneration of glutamatergic neurons in the cerebral cortex andhippocampal formation and of cholinergic neurons in the basal forebrain,extracellular deposit of amyloid plaque, and intracellularneurofibrillary tangles are pathological features of AD. In AD, theproduction of lipid peroxidation, 8-hydroxy deoxyguanosine, proteincarbonyls, nitration, or oxidative crosslinking of proteins by excessgeneration of free radicals has been reported, suggesting that oxidativestress plays a causative role in neuronal death in AD [Vitek et al.,Proc. Natl. Acad. Sci. U.S.A., 91:4766-4770 (1994); Smith et al.,Trends.Neurosci., 18:172-176 (1995), Mol.Chem.Neuropathol., 28:41-48(1996), Proc. Natl. Acad. Sci. U.S.A., 94:9866-9868 (1997); Montine etal., J. Neuropathol. Exp. Neurol., 55:202-210 (1996)]. As a matter offact, the therapeutic effects of anti-oxidants have been extensivelyinvestigated in AD patients. Zn²⁺ is accumulated in the brain (amygdala,hippocampus, inferior parietal lobule, superior and middle temporalgyri) of AD patients, mainly in the center and surround of amyloidplaque and induces aggregation of beta amyloid [Bush et al., Science265:1464-1467 (1994); Lovell et al., J. Neurol. Sci., 158:47-52 (1998)].Therefore, the compounds in the present invention showing protectiveeffect against oxidative stress and Zn²⁺ toxicity can be used astherapeutic drugs for AD.

APPLICATION EXAMPLE 8 Ocular Diseases and Cataract

In glaucoma, the increased intraocular pressure blocks blood flow intoretina, causes retinal ischemia, and induces excessive release ofglutamate into synaptic cleft. Once released, glutamate induces NMDAreceptor-mediated excitotoxicity by opening calcium channels andincreasing intracellular Ca²⁺ concentration in post-synaptic neurons.The degeneration of retina cells can also occur through the increasedgeneration of reactive oxygen species during reperfusion [Osborne N. N.et al., Surv. Opthalmol., 43 suppl., 1:S102-28 (1999); Hartwick A. T.,Optom. Vis. Sci., 78:85-94 (2001)]. Administration of NMDA receptorantagonists or anti-oxidants inhibits retinal degeneration in animalmodel of glaucoma [Gu. Z. et al., Nippon Ganka Gakkai Zasshi, 104:11-6(2000); Vorwerk C. K. et al., Surv Ophthalmol., 43 suppl., 1:S142-50(1999); Schwartz M. et al., Eur. J. Ophthalmol., 9 Suppl., 1:S9-11(1999)].

In cases of retinopathy and macular degeneration, neuronal degenerationcan be blocked by inhibition of excitotoxicity and oxidative stress,main causes of these diseases [Lieth E. et al., Clin. ExperimentOphthalmol., 28(1):3-8 (2000); Moor P. et al., Exp. Eye Res., 73:45-57(2001); Winkler B. S. et al., Mol. Vis., 5:32 (1999); Simonelli F. etal., Clin. Chim. Acta., 320:111-5 (2002)].

Cataract is a senile disease accompanying lenticular opacity. Oxidativestress has been considered as a primary mediator of cataract. In fact,anti-oxidants have been applied to treat cataract [Varma et al., Curr.Eye Res. 3:35-57(1984); Anderson et al., Adv. Exp. Med. Biol.,366:73-86(1994)].

Therefore, the compounds in the present invention showing multipleprotective effects against excitotoxicity and oxidative stress can beused as therapeutic drugs for ocular diseases.

APPLICATION EXAMPLE 9 Drug Addiction

The activation of mesolimbic dopaminergic neurons in the ventraltagmental area that project to the nucleus accumbens is essential forthe process of drug addition that requires neuronal excitability [SelfD. W. and Nestler E. J., Annu. Rev. Neurosci., 18:463-95 (1995)].Several lines of evidence supports that NMDA receptor antagonists can beapplied to reduce neuronal adaptability to abused drugs and have beensuggested as new therapeutic agents for drug addiction [Boening et al.,Alcohol Clin. Exp. Res., 25:127S-131S (2001); Vorel et al., Science,292:1175-8 (2001)].

APPLICATION EXAMPLE 10 Depression

Tricyclic antidepressants and MAO (monoamine oxidase) inhibitors,antidepressants, increase neurotransmission of noradrenaline andserotonin. The existing therapeutic drugs induce a lot of side effectsthrough interacting with other nervous system and have no therapeuticeffect in 30% of depressed patients [Pacher et al., Curr. Med. Chem.,February 8 (2):89-100 (2001)]. Recently, NMDA receptor antagonists wereshown to be applicable as new therapeutic drugs for depression [Le D. A.and Lipton S. A., Drugs Aging, 18:717-724 (2001); Petrie et al.,Pharmacol. Ther., 87:11-25 (2000)].

APPLICATION EXAMPLE 11 Pain

Neuropathic pain results from the increased neurotransmission followingperipheral injury and neural tissues damage in relation with surgery,cancer patients, and trauma etc [Hempenstall K. and Rice A. S., Curr.Opin. Investig. Drugs., March;3 (3):441-8 (2002); McDonnell et al.,Curr. Oncol. Rep., 2:351-7 (2000)]. Since the activation of NMDAreceptor is necessary for the processing of neuropathic pain, it hasbeen reported that NMDA receptor antagonists can be used as therapeuticdrugs to treat neuropathic pain [Parson. C. G., Eur. J. Pharmacol.,429:71-8 (2001); Hewitt, Clin. J. Pain., 16:S73-9 (2000)].

APPLICATION EXAMPLE 12 Multiple Sclerosis, Meningitis, Encephalitis orHydrocephalus

Oxidative stress plays a role in the pathogenesis of multiple sclerosis,meningitis, encephalitis, and hydrocephalus. Levels of anti-oxidantssuch as retinal, a-tocopherol, b-carotene and ascorbic acid are reducedin the body of multiple sclerosis patients [Calabrese, V et al., Int. J.Clin. Pharmacol. Res., 14(4):119-123(1994); Besler H. T. et al., Nutr.Neurosci. 5(3):215-220(2002); Nutr. Neurosci. 6(3):189-196(2002)].Increased ROS generation and neuronal cell death are observed atmeningitis patient and its animal model [Maurizi C. P., Med. Hypotheses,52(1):85-87(1999); Christen S. et al., Free Radic. Biol. Med,31(6):754-762(2001); Kastenbauer S. et al., Neurology,58(2)186-191(2002)], infection model by encephalitis virus [Fujii, S. etal., Virology, 256(2):203-212(1999); Raung S. L. et al., Neurosci.Lett., 315(1-2):9-12(2001)], and hydrocephalus patients [Nuss J. I. etal., Am. J. Vet Res., 28(127):1909-1913(1967.); Vannucci R. C. et al.,Dev. Med. Child. Neurol., 22(3):308-316(1980); Radwanska-Wala B. et al.,Pathol. Res. Pract., 198(6):421-423(2002)]. Therefore, the compounds inthe present invention showing protective effect against oxidative stresscan be used as therapeutic drugs for neuronal death induced by multiplesclerosis, meningitis, encephalitis or hydrocephalus.

The present invention has been described in an illustrative manner, andit is to be understood that the terminology used is intended to be inthe nature of description rather than of limitation. Many modificationsand variations of the present invention are possible in light of theabove teachings. Therefore, it is to be understood that within the scopeof the appended claims, the invention may be practiced otherwise than asspecifically described.

INDUSTRIAL APPLICABILITY

As described hereinbefore, Tetrafluorobenzyl derivatives orpharmaceutically-acceptable salts, and pharmaceutical compositioncontaining the same as the effective component can prevent and treat theneurodegenerative diseases such as amyotrophic lateral sclerosis,Parkinson's diseases, Huntington's disease or Alzheimer's disease, theconvulsive neuronal diseases such as epilepsy etc, and brain injury bystroke, trauma, or hydrocephalus, ocular diseases by glaucoma andretinopathy, mental diseases by drug addiction and depression,neuropathic pain, inflammatory diseases by meningitis and encephalitisas described above.

1. A method for inhibiting N-methyl-D-aspartate (NMDA) receptor,comprising administering to a patient in need thereof atetrafluorobenzyl derivative or its pharmaceutically acceptable salt,wherein the tetrafluorobenzyl derivative is represented by the followingchemical formula:

wherein, R₁, R₂ and R₃ are hydrogen or halogen, R₄ is hydroxy, alkyl,alkoxy, halogen, alkoxy substituted with halogen, alkanoyloxy or nitro,and R₅ is carboxyl acid, C(═O)—O—C₁-C₄ alkyl, carboxyamide, sulfonicacid, halogen or nitro; or a pharmaceutically acceptable salt thereof.2. The method according to claim 1 wherein the tetrafluorobenzylderivative is where R₁, R₂ and R₃ are halogen.
 3. The method accordingto claim 2 wherein the tetrafluorobenzyl derivative is where halogen isF.
 4. The method according to claim 1 wherein the tetrafluorobenzylderivative is where R₄ is hydroxy or alkoxy.
 5. The method according toclaim 1 wherein the tetrafluorobenzyl derivative is where R₄ is hydroxy.6. The method according to claim 1 wherein the tetrafluorobenzylderivative is where R₅ is carboxyl acid or C(═O)—O—C₁-C₄ alkyl.
 7. Themethod according to claim 1 wherein the tetrafluorobenzyl derivative iswhere R₅ is carboxyl acid.
 8. The method according to claim 1 whereinthe tetrafluorobenzyl derivative is where R₁, R₂ and R₃ are halogen, R₄is hydroxy or alkoxy, and R₅ is carboxyl acid, or a pharmaceuticallyacceptable salt thereof.
 9. The method according to claim 8 wherein thetetrafluorobenzyl derivative is where R₄ is hydroxy.
 10. The methodaccording to claim 8 wherein the tetrafluorobenzyl derivative is wherehalogen is F.
 11. The method according to claim 1 wherein thetetrafluorobenzyl derivative is2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoicacid or a pharmaceutically acceptable salt thereof.
 12. The methodaccording to claim 11 wherein the tetrafluorobenzyl derivative is2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoicacid.
 13. The method according to claim 11 wherein the tetrafluorobenzylderivative is a pharmaceutically acceptable salt of2-hydroxy-5-(2,3,5,6-tetrafluoro-4-trifluoromethyl-benzylamino)-benzoicacid.
 14. The method according to any one of claims 1-13, additionallyincluding a pharmaceutically acceptable carrier, diluent or excipient.